The pod borer (Helicoverpa armigera) is one of the most serious pest of chickpea and plant

resistance is an important component for managing this pest. To develop cultivars with

resistance to insects, it is important to understand the role of different components

associated with resistance to insects. Therefore, in this study we characterized RIL’s

(recombinant inbred lines) population for total phenol content leaves and organic acid

profiles in the leaf exudates which are associated with tolerance to H. armigera. Chickpea

leaves contained phenol and five major organic acids, which were identified as malic acid,

oxalic acid, acetic acid, citric acid, and fumaric acid. The high performance liquid

chromatography (HPLC) profiles of the leaf exudates of 196 RIL’s exhibited amounts of all

organic acids were negatively correlated with egg count, larval incidence and with pod

damage. Total phenol levels were negatively associated with egg count, larval incidence

and pod damage percentage

Akhare, A A

Bangar, H A

Bangar, S S

Dudhare, M S

Gahukar, S J

Sharma, H C

Wadaskar, R M

ICRISAT Open Access Repository

Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229   223    Original Research Article  Expression of Tolerance to Pod Borer, Helicoverpa armigera (Lepidoptera: Noctuidae) in Relation to Biochemical Content of Chickpea Leaves  S.S. Bangar1, H.A. Wagh1, M.S. Dudhare1, S.J. Gahukar1*, R.M. Wadaskar1,  A.A. Akhare1 and H.C. Sharma2  1Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra-444 104, India 2International Crops Research Institute for the Semi-Arid Tropics, Patancheru, Andhra Pradesh-502 324, India *Corresponding author                              A B S T R A C T                          Introduction  Chickpea (Cicer arietinum L.) is one of the important grain legume crops of India which plays an important role in food security and balanced diet and is virtually an indispensable item in the kitchen (Bhatt and Patel, 2001). Pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a key pest of chickpea and accounts about 90 to 95 % of the total damage caused by all the insect pests (Sachan and Katti. 1994). The damage caused by this pest on chickpea ranges up to 10% to 60% different farming systems (Vaishampayam and Veda, 1980). In Maharashtra 60-80% crop losses are       reported due to this pest from early vegetative to podding stage in chickpea (Patil et al., 2007).   Low to moderate levels of resistance to H. armigera have been identified in the chickpea germplasm (Dias et al., 1983; Lateef, 1985; Lateef and Sachn, 1990).   Total phenol content and organic acids in leaf exudates produced by the trichomes on the surface of chickpea plants, of which malic acid and oxalic acid are the principal components; result in oviposition non-preference and anti-feedant effects on H. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Special Issue-6 pp. 223-229 Journal homepage: http://www.ijcmas.com  The pod borer (Helicoverpa armigera) is one of the most serious pest of chickpea and plant resistance is an important component for managing this pest. To develop cultivars with resistance to insects, it is important to understand the role of different components associated with resistance to insects. Therefore, in this study we characterized RIL’s (recombinant inbred lines) population for total phenol content leaves and organic acid profiles in the leaf exudates which are associated with tolerance to H. armigera. Chickpea leaves contained phenol and five major organic acids, which were identified as malic acid, oxalic acid, acetic acid, citric acid, and fumaric acid. The high performance liquid chromatography (HPLC) profiles of the leaf exudates of 196 RIL’s exhibited amounts of all organic acids were negatively correlated with egg count, larval incidence and with pod damage. Total phenol levels were negatively associated with egg count, larval incidence and pod damage percentage. K e y w o r d s   Helicoverpa armigera, RIL’s, Chickpea, HPLC, Resistance, Organic acids, Total phenol.     Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229 224  armigera (Rembold et al., 1990; Yoshida et al., 1995). The present studies focused on estimation of total phenol and acid exudates in the leaf samples of recombinant inbred lines of chickpea developed from crossing ICC 506 EB (resistant to pod borer) X Vijay (susceptible to pod borer) to study the association of total phenol and organic acids with resistance to H. armigera.  Materials and Methods  Plant material  The early maturing pod borer resistant genotype ICC 506 EB and pod borer susceptible genotype Vijay were used as parents. Homozygous population consisting of 196 RIL’s developed from cross between selected parents was used for present study.  Field trails  196 RIL’s along with parents were evaluated in field experiments at Dr. PDKV, Akola. The site is located in Vidarbha region of Maharashtra state of India with substantial chickpea production and natural occurrence of pod borer.   The experiment was conducted in RBD with three replications. Row to row distance maintained was 30 cm while plant to plant distance was 10 cm. Number of eggs and larvae were counted during the vegetative (15 DAE), flowering (45 DAE) and pod formation (60 DAE) stages of the crop.   Pod damage by H. armigera larvae was quantified by expressing the number of pods bored as a percentage to the total number of pods. Ten tagged plants were harvested individually and average yield was taken as yield per plant in each plot. All the observations were recorded on 10 tagged plants from each RIL at random. Total phenol estimation  Total phenol content of leaves was estimated at flowering stage of crop. Total phenols estimation was carried out with Folin-Ciocaletu reagent (FCR). Phenols react with an oxidizing agent phosphomolybdate in Folin–Ciocalteu reagent under alkaline conditions and result in the formation of a blue coloured complex, the molybdenum blue which was measured at 650nm using spectrophotometer (Bray and Thorpe, 1954).  Organic acids estimation  Chickpea plants grown in the greenhouse were used for collection of acid exudates. Glass vials of 15 ml capacity were used for collecting the acid exudates. The weight of the vial along with 5 ml of distilled water was recorded (W1), and then ten fully expanded leaflets were collected from each genotype at the flowering stage and placed in the vials. The weight of the vial + leaves was recorded (W2), and fresh weight of the leaves was computed by subtracting W1 from W2. The vials were Vortexed for 1 min. The water-extracted chemicals were filtered through 0.45 µ Millipore filter, and 2 ml of extract was taken into a screw top vial (12  32 mm) with an injection needle. The contents were sonicated for 10 min for dissolving the solutes and degassing of solvents, and then used for HPLC analysis. The HPLC (Waters 2695 Separation Module with photodiode detector) running parameters were as follows. Mobile phase consisted of 25 mM KH2PO4 pH 2.5. Flow rate 0.8ml min-1, Run time 20 min per sample. Injected sample volume was 20µl. Three samples of each test genotypes were run through the HPLC to obtain as estimate of the organic acids present in water-soluble leaf exudates of different chickpea genotypes. Standard samples of known organic acids (oxalic, malic, citric, fumaric, Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229 225  and acetic acids) were used to spike the HPLC peaks to identify different acids. After identification of peaks corresponding to different organic acids, a range of concentrations for each organic acid were run through the HPLC to obtain a normal curve. The amounts of different organic acids present in the leaves of different chickpea genotypes were estimated from normal curves based on peak areas.  Statistical analysis  For all the phenotypic observations recorded the data were subjected to analysis of variance by using WINDOSTAT release 5.2. The significance of differences between the treatments was measured by F- test at P = 0.05, whereas the treatment means were compared using the least significant difference (LSD) at P = 0.05. Phenotypic correlation coefficients were estimated using the formula of Singh and Choudhary, (1996).  Results and Discussion  The present study involved 196 recombinant inbred lines (RILs) and two parents. RIL population was derived from the cross ICC 506 EB (resistant) X Vijay (susceptible). The resistant parent ICC 506 EB showed minimum egg and larval count at all stages of observation during both years, Less % pod damage (6.19, 6.58), pod damage score (1.00, 1.00), while high yield per plant (9.97, 10.85) was recorded during two subsequent years respectively. On the contrary, susceptible parent Vijay showed higher egg and larval count at all stages of observation during both years when compared with ICC 506 EB; Higher percent pod damage (17.37, 15.32), pod damage score (2.00, 1.75) was recorded, while less yield (6.34, 6.14) was recorded during years 2010 and 2011 respectively. Range of phenotypic value of pod borer resistance component traits in mapping population of RIL’s is given in Table 1.  Higher total phenol content (44.71 µg/100 g of leaves) was recorded in ICC 506 EB than Vijay, also higher amounts of all organic acids viz. oxalic (57.31), acetic (103.91), fumaric (1.88) except malic (13.12) and citric acid (0.00) was recorded than Vijay.   While less total phenol content (33.97 µg/100g of leaves) was recorded than ICC 506 EB, also lower amounts of all organic acids (µg/g of dried leaves) viz. oxalic (15.14), acetic (0.00), fumaric (0.70) except malic (150.64) and citric acid (47.47) was recorded in comparison with ICC 506 EB..  Total phenol was ranged between 34.29 and 48.40. Lowest total phenol was observed in RIL number 136 while highest was observed in 85. In case of different organic acids viz. oxalic acid, malic acid, acetic acid, citric acid, fumaric acid (µg/gm of dried leaf) was ranged between 1.03 to 172.36, 0.00 to 869.04, 0.00 to 57.26, 0.00 to 111.43, and 0.00 to 9.20 respectively. Highest oxalic, malic, acetic, citric, fumaric acids was found in RIL number 10, 41, 76, 87 and 102 respectively; while lower concentrations of oxalic, malic, acetic, citric, fumaric acid was observed in 88, 134, 135, 151, and 179 number RIL (Fig. 1).  Resistance/tolerance pod borer is a complex character and it is controlled by many factors. For effective selection to improve resistance, it is necessary to have an understanding of various associated traits and nature of their association with host plant resistance. Association analysis employed in this study provides such required information. In present study total phenol content exhibited significant negative correlation with percent pod damage (-0.15) Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229 226  also all organic acids exhibited negative association with percent pod damage (Table 2). Similar results were obtained for phenols and malic acid in chickpea (Shahapur, 1997).  The lines with less percent pod damage showed significantly higher total phenols compared to susceptible lines. In pigeonpea also, low amino acid, protein and sugar content and high phenol content induce resistance against pod borer (Sahoo and Patnaik, 2003). Low acidity of the chickpea leaf extracts has earlier been reported to be associated with susceptibility to H. armigera (Rembold et al., 1990; Rembold, 1981; Rembold et al., 1989; Srivastava and Srivastava, 1989). However, resistance expressed ICC 506 has been attributed to factors other than acidity (Patnaik and Senapati, 1985). Malic acid and oxalic acid in the acid exudates are known to play a considerable role in genotypic susceptibility to H. armigera.   Antifeedant and/or antibiotic properties of organic acids may influence the host selection and feeding behavior, and thus, influence the growth and development of H. armigera larvae and determine the extent of damage on a particular genotype (Rembold et al., 1990; Rembold and Winter, 1982). The present studies indicated that in addition to oxalic acid and malic acid, citric acid, acetic acid, and fumaric acid also play an important role on genotypic resistance to H. armigera. Monitoring the amounts of organic acids through HPLC can be used to select chickpea genotypes for resistance to H. armigera.  Table.1 Means of parents ICC 506 EB and Vijay, the RIL’s derived from their cross for egg count, larval count, percent pod damage, yield per plant and biochemical parameters   Range of RIL’s across two environments Mean of RIL’s Vijay ICC 506 EB SE ±  Min Max    Egg count at 15 DAS 0.50  6.17 2.33 7.50 2.67 0.20 Larval count at 15 DAS 2.67 8.33 4.72 5.50 3.00 0.16 Egg count at 45 DAS 0.50 10.00 2.90 3.00 0.83 0.22 Larval count at 45 DAS 0.33 9.83 3.43 6.17 1.00 0.21 Egg count at 60 DAS 0.83 9.00 4.52 7.00 0.83 0.21 Larval count at 60 DAS 2.33 10.17 5.74 9.50 1.83 0.21 % Pod damage 9.76 27.94 17.18 6.38 16.34 0.20 Yield per plant (g) 3.40 9.18 6.24 10.41 6.24 0.17 Total phenol (mg/100g of leaf sample) 34.29 48.40 42.34 33.97 44.71 0.14 Oxalic acid (mg/100g of leaf sample) 1.03  172.36 44.12 15.14 57.31 0.21 Malic acid (mg/100g of leaf sample) 0.00 869.04 165.58 150.64 103.91 0.21 Acetic acid (mg/100g of leaf sample) 0.00 57.26 3.32 0.00 13.12 0.05 Citric acid (mg/100g of leaf sample) 0.00 111.43 30.13 47.47 0.00 0.09 Fumaric acid (mg/100g of leaf sample) 0.00 9.20 1.31 0.70 1.88 0.05 Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229 227  Table.2 Correlation coefficients for components of resistance to pod borer  * – Significant at p = 0.05, ** – Significant at p = 0.01   Total phenol Oxalic Acid Malic Acid Acetic Acid Citric Acid Fumaric Acid Egg count at 15 DAS Larval count at 15 DAS Egg count at 45 DAS Larval count at 45 DAS Egg count at 60 DAS Larval count at 60 DAS Yield  % Pod damage Total phenol 1.00 0.31** 0.16* -0.12 -0.14* -0.05 -0.05 -0.13 0.03 -0.07 -0.03 -0.06 0.01 -0.15* Oxalic Acid  1.00 0.04 -0.30** -0.35** -0.14* -0.05 -0.02 -0.14* -0.10 0.17** -0.14* -0.10 -0.09 Malic Acid   1.00 -0.14* 0.13 -0.05 0.09 -0.05 0.15* -0.17** -0.19** -0.20** -0.07 -0.11 Acetic Acid    1.00 0.01 0.01 -0.02 0.04 -0.14* -0.05 -0.05 -0.04 -0.02 -0.14* Citric Acid     1.00 0.01 -0.11 0.02 -0.08 -0.25** -0.04 -0.07 0.04 -0.01 Fumaric Acid      1.00 -0.05 -0.04 -0.11 -0.03 -0.09 -0.10 0.06 -0.02 Egg count at 15 DAS       1.00 -0.01 -0.10 0.01 -0.05 0.11 -0.04 0.06 Larval count at 15 DAS        1.00 0.04 0.09 0.07 0.26** 0.09 0.10 Egg count at 15 DAS         1.00 0.28** 0.74** 0.75** 0.02 -0.05 Larval count at 15 DAS          1.00 0.68** 0.78** -0.10 0.03 Egg count at 15 DAS           1.00 0.93** -0.17** -0.01 Larval count at 15 DAS            1.00 -0.09 0.02 Yield              1.00 -0.26**  % Pod damage              1.00 Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 223-229 228  Fig.1 (A to F) Frequency distribution of 196 RIL’s for biochemical components of resistance viz., total phenol, oxalic, malic, acetic, citric and fumaric acid content of leaves   1A      1B  1C      1D  1E      1F  References  Bhatt N. 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Expression of Tolerance to Pod Borer, Helicoverpa armigera (Lepidoptera: Noctuidae) in Relation to Biochemical Content of Chickpea Leaves

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Expression of Tolerance to Pod Borer, Helicoverpa armigera (Lepidoptera: Noctuidae) in Relation to Biochemical Content of Chickpea Leaves

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